Classical swine fever (CSF) or hog cholera is a highly contagious and often fatal disease of pigs which is characterised by fever and haemorrhages and can run an acute or chronic course (Van Oirschot. 1986. Hog cholera, p. 289-300. In Diseases of Swine. Iowa State University Press, Ames). Outbreaks of the disease occur intermittently in several European and other countries and can cause large economic losses.
Vaccination of pigs with a live attenuated Classical swine fever virus (CSFV) vaccine strain, the "Chinese" strain (C-strain), protects pigs against CSF (Terpstra and Wensvoort. 1988. Vet. Microbiol. 16: 123-128). A major drawback of vaccinating pigs with the conventional vaccines, of which the C-strain is one, is that these vaccinated pigs cannot be distinguished serologically from pigs infected with a CSFV field strain. The C-strain, however, is considered one of the most effective and safe live vaccines. Addition of a (serological) marker to the C-strain would be highly advantageous and would improve the vaccine.
CSFV is a member of the Pestivirus genus of the Flaviviridae (Francki, R. I. B. et al. 1991. Flaviviridae, p. 223-233. In Fifth report of the International Committee on Taxonomy of Viruses. Archiv. Virol. Suppl. 2, Springer Verlag, Vienna). The other two members of the Pestivirus genus, which are structurally, antigenically and genetically closely related to CSFV, are Bovine viral diarrhoea virus (BVDV) mainly affecting cattle, and Border disease virus (BDV) mainly affecting sheep (Moennig and Plagemann, 1992. Adv. Virus Res. 41: 53-98; Moormann et al., 1990. Virology 177: 184-198; Becher et al. 1994. Virology 198: 542-551).
The genomes of pestiviruses consist of a positive strand RNA molecule of about 12.5 kb (Renard et al. 1985. DNA 4: 429-438; Moormann and Hulst 1988. Virus Res. 11: 281-291; Becher et al. 1994. Virology 198: 542-551). The positive strand RNA genomes of several non-cytopathogenic BVDV strains, however, may be considerably larger (Meyers et al. 1991. Virology 180: 602-616; Meyers et al. 1992. Virology 191: 368-386; Qi et al. 1992. Virology 189: 285-292).
An inherent property of viruses with a positive strand RNA genome is that their genomic RNA is infectious, i.e. after transfection of this RNA in cells that support viral replication infectious virus is produced. As expected, the genomic (viral) RNA of pestiviruses is also infectious (Moennig and Plagemann, 1992. Adv. Virus Res. 41: 53-98).
For several years recombinant DNA technology has allowed in vitro transcription of cloned DNA. This possibility has opened the way to synthesize infectious RNA in vitro from a DNA copy of the genome of a positive strand RNA virus. It is well known in the field of molecular engineering that DNA, in contrast to RNA, is easily manipulated by site directed mutagenesis. Hence, the availability of the technique to produce synthetic infectious RNA has greatly enhanced the study of e.g. replication, virulence, pathogenesis, RNA recombination, vector development, and antiviral strategies of the positive strand RNA viruses. However, application of the technology may cause severe problems. The nature of these problems has been described in a recent review by Boyer and Haenni. 1994. (Virology 198: 415-426). In fact, the success or failure to construct a full-length DNA copy of the genome of a positive strand RNA virus and to produce synthetic infectious RNA from such a full-length DNA copy cannot be reliably predicted.